Influence of heat stress or feed restriction on plasma progesterone ...

Livestock Production Science 68 (2001) 231–241 www.elsevier.com / locate / livprodsci

Influence of heat stress or feed restriction on plasma progesterone, oestradiol-17b, LH, FSH, prolactin and cortisol in Holstein heifers a, b c a a B. Ronchi *, G. Stradaioli , A. Verini Supplizi , U. Bernabucci , N. Lacetera , P.A. Accorsi d , A. Nardone a , E. Seren d a

Institute of Animal Husbandry, University of Tuscia, via C. de Lellis, 01100 Viterbo, Italy Department of Animal Production Science, University of Udine, via delle Scienze 208, 33100 Udine, Italy c Institute of Animal Production, University of Perugia, via San Costanzo 4, 06126 Perugia, Italy d Department of Veterinary Morphophysiology and Animal Production, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia ( BO), Italy b

Received 7 July 1999; received in revised form 15 February 2000; accepted 25 July 2000

Abstract The aim of the study was to compare the effects of heat stress and feed restriction on hormonal secretion (progesterone, oestradiol-17b, luteinizing hormone, follicle-stimulating hormone, prolactin and cortisol) in Holstein heifers. Ten pubertal heifers were divided into two groups of five (A and B) and housed in climatic chambers. After a pre-experimental period, the heifers were synchronised for oestrus and monitored for three (group B) or four (group A) consecutive oestrus cycles (OC). In the first OC, both groups were maintained under thermal comfort (TC) and fed on an ad libitum basis. In the second OC, group A was maintained under TC whereas group B was exposed to high air temperatures (HAT); both groups were fed on an ad libitum basis. In the third OC and until day 17 of the fourth OC, group A was kept under TC and fed a restricted diet (the same ration ingested by group B under HAT). At the end of HAT exposure, group B was removed from the study. Exposure to HAT caused development of ovarian cysts in two heifers, an increase in plasma concentrations of prolactin, a decrease in concentrations of cortisol and progesterone, and a 23% reduction in dry matter intake. Feed restriction did not modify any of the parameters considered. Results of this study indicated that the effects of HAT on the above parameters are not altered by a reduction in feed intake.  2001 Elsevier Science B.V. All rights reserved. Keywords: Dairy cattle; Heat stress; Feed restriction; Reproduction; Hormonal secretion

1. Introduction Several authors have documented depression of *Corresponding author. Tel.: 1 39-761-357-444; fax: 1 39761-357-434. E-mail address: [email protected] (B. Ronchi).

fertility in dairy cows during hot seasons and a decline in conception rate (Ron et al., 1984; Wise et al., 1988), an increase in embryo mortality (Ryan et al., 1993), impaired luteal function (Wolfenson et al., 1993; Howell et al., 1994), and disturbances in gonadotrophin (Gilad et al., 1993) and oestradiol secretion (Wilson et al., 1998) have been observed in

0301-6226 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 00 )00232-3

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B. Ronchi et al. / Livestock Production Science 68 (2001) 231 – 241

heat-stressed cattle. Moreover, high air temperatures are known to modify oestrus expression (Gwazdauskas et al., 1981), increase frequency of silent oestrus (Rodtian et al., 1996) and change follicular dynamics (Stradaioli et al., 1994; Wilson et al., 1998). The effects of heat stress on reproductive function and hormone changes (Richards et al., 1995) have been thought to be partly related to a reduction in feed intake. The main purpose of this study was to investigate the common and individual effects of heat stress and feed restriction on ovarian (progesterone, oestradiol-17b) and pituitary (luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL)) functions, and on cortisol secretion in Holstein heifers.

2. Materials and methods

2.1. Animals, experimental design and feeding The study was carried out using 10 cyclic 15- to 16-month-old Holstein heifers (two pairs of full-sibs and three pairs of half-sibs). The animals were

assigned to two experimental groups (A and B) which were arranged by splitting each of the five pairs of sibs. The two groups were similar in body weight (426.6668.6 vs. 420.6669.3 kg for groups A and B, respectively, P . 0.05) and body condition score (2.560.4 vs. 2.660.5 for groups A and B, respectively, P . 0.05). Body condition was scored according to Edmondson et al. (1989) and always by the same person. The heifers were housed in two climatic chambers with individual tie stalls. Each chamber was 5.6 m wide, 10.0 m long and had a 162.4 m 3 capacity. The two chambers, each capable of housing a maximum of six mature cows, were equipped with individual feeders and water bowls. Ambient temperature and relative humidity were computer-controlled using heater and refrigerator units which were monitored continuously. The photoperiod schedule (14 h light (400 lux) and 10 h dark) and air circulation (0.5 ambient volume / h) were maintained during the entire trial. The schematic representation of the experimental design is shown in Fig. 1. The heifers were maintained for a 14-day pre-experimental period under thermal comfort (TC) (188C and 70% relative

Fig. 1. Schematic representation of the experimental design.


humidity 5 temperature–humidity index (THI) of 65) to allow them to adapt to the new housing conditions. During that period no data were collected. The experimental period lasted for 95 days and started immediately after the end of the pre-experimental period. At the end of the pre-experimental period, the heifers of both groups (A and B) were synchronised with 3 mg of norgestomet (Crestar, Intervet, Italy) which were ear implanted for 9 days. Seven days after implantation, 0.5 mg of cloprostenol (Estrumate, Pitman-Moore, Italy) was injected i.m. Within 72 h of implant removal, all animals showed signs of oestrus (behavioural (bellowing) and physical (red and swollen vulva, and clear mucus discharge from vulva)). From the first oestrus (1stO), all heifers were monitored twice daily, in the morning and in the evening, until the end of the trial to detect the signs of the subsequent oestrus. The two groups were maintained under TC for the first 17 days of the first oestrus cycle (OC). That period was considered as a covariate period. Starting on day 17 of the first OC, after morning blood sampling, heifers in group B were exposed to high air temperatures (HAT) (328C and 70% relative humidity 5 THI of 84) while group A remained under TC. A THI of 84 was chosen in order to induce a condition of intermediate heat stress (between mild and severe). The passage from TC to HAT took 24 h to complete. The HAT treatment for group B started 4 days before the expected second oestrus (2ndO) and ended when all heifers ovulated in the third oestrus (3rdO) (Fig. 1). After this ovulation, the heifers kept under HAT were removed from the study. After ovulation occurred in the 3rdO, feed was restricted for group A heifers (they were given the same ration as that which group B heifers voluntarily ingested under HAT (see Section 3)) and they remained under TC (TCRF). The TCRF treatment was maintained for group A during the third OC and until day 17 of the fourth OC (Fig. 1). During the third OC, no measurements were carried out. The diet consisted of Italian ryegrass hay and commercial concentrate (82 / 18 ratio on a dry matter (DM) basis). The ration was administered twice daily (at 09:00 and at 17:00 h) and contained 0.62 fodder units for milk production / kg and 12% crude protein on a DM basis. Individual feed refusals were

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weighed once daily at 08:30 h and feed intake was recorded. The diet was adjusted continuously to maintain a constant forage / concentrate ratio. The heifers had free access to tap water. Individual water intake was recorded once daily at 08:30 h using water meters attached to the water bowls.

2.2. Rectal temperatures and blood samples Rectal temperatures (RT) were recorded at 09:00 h every 2 to 3 days using a glass precision thermometer with a prismatic section (Artsana Veterinaria, Como, Italy) and 0.18C accuracy. Blood (20 ml) was collected from the jugular vein every 15 min for 12 h (from 08:00 to 20:00 h) on day 0 (oestrus), and on day 12 of the 1st (covariate period), 2nd (TC and HAT treatments) and 4th (TCRF treatment) OC (Fig. 1) to determine plasma LH, FSH and PRL. The day before frequent blood sampling, all heifers were fitted with a jugular catheter (14-gauge, Intraflon 2, Vygon, France). In TC and HAT treatments during the 3rdO, blood samples (10 ml) were collected, at 3-h intervals, from the first sign of oestrus until ovulation, which was monitored at 6-h intervals using a real time array ultrasound scanner (Toshiba Sonolyer, SAL32A, Japan) equipped with a 5 MHz intrarectal probe. Blood samples were utilised to determine plasma LH, oestradiol-17b and cortisol. Blood samples (10 ml) were also taken every 2 to 3 days from day 0 until day 17 of the 1st, 2nd and 4th OC at 08:00 h (Fig. 1) to determine plasma progesterone, oestradiol-17b, PRL and cortisol. Blood samples were collected from the jugular vein into heparinised evacuated tubes and immediately centrifuged at 2000 3 g for 10 min. Plasma was stored at 2 208C until analysis.

2.3. Hormone assays Progesterone and oestradiol-17b were determined by specific radioimmunoassays (RIA). The sensitivity (90% B / B o ) of the progesterone antibody was 2.5 ng / ml and the cross-reactivities were: 9.7% with hydroxyprogesterone-11a, 1.5% with hydroxyprogesterone-17a, 0.3% with hydroxyprogesterone-20a, , 0.002% with testosterone, 0.05% with cortisol, , 0.0002% with oestrone

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and , 0.0001% with oestradiol-17b. The sensitivity (90% B / B o ) of the oestrogen antibody was 1.01 pg / ml and the cross-reactivities were: 100% with oestradiol-17b, 1.5% with oestradiol-17a, 0.8% with oestriol, 0.03% with oestriol sulphate, 1.5% with oestrone and 0.1% with oestrone sulphate. Plasma LH concentrations were determined by a heterologous double-antibody method. This involves the use of rabbit antiserum to bovine LH (NIH-LHB10) at a final dilution of 1:200,000, which displayed negligible cross-reactivity with adrenocorticotrophin hormone (ACTH), FSH, growth hormone (GH), PRL and thyroid-stimulating hormone (TSH). The balance point (the concentration of hormone that induces a displacement of 50% B / B o ) was 540 pg / tube and sensitivity (90% B / B o ) was 55 pg / tube. NIH-LH-B10 was used as the standard reference and bovine LH LER 1072-2 was used to prepare the radio-iodinated tracer according to Salacinski et al. (1981). The intra-assay and inter-assay coefficients of variation (CV) were 9.7% and 13.8%, respectively. A heterologous double-antibody RIA system, using rabbit antiserum to ovine FSH (NIDDK antioFSH-I) at a final dilution of 1:40,000, was utilised for the measurement of plasma FSH. The crossreactivity with ACTH, PRL, GH and LH was negligible ( , 0.003%). USDA-bFSH-I-2 was used as the standard reference and to prepare the radioiodinated tracer according to Salacinski et al. (1981). The balance point was 164 pg / tube and sensitivity (90% B / B o ) was 27 pg / tube. The intra-assay and inter-assay CV were 8.1% and 12.7%, respectively. Plasma prolactin (PRL) concentrations were measured by a heterologous double-antibody RIA, using a rabbit anti-oPRL serum at a final dilution of 1:300,000. LER-891-bPRL was used as a standard reference and NIDDK-bPRL-I-1 was used to prepare the radio-iodinated tracer according to Salacinski et al. (1981). The balance point was 322 pg / tube and the sensitivity (90% B / B o ) was 33 pg / tube. The intra-assay and inter-assay CV were 6.9% and 14.7%, respectively. Plasma cortisol concentrations were evaluated using a RIA. The sensitivity (90% B / B o ) of the cortisol antibody was 4.93 ng / ml, and the cross-reactivities were: 20.4% with cortisone, 74.6% with deoxycortisol-11a, 1.13% with corticosterone, and 0% with progesterone and oestrogens.

2.4. Statistical analysis Each value of LH, FSH and PRL which was 30% greater than the previous value and was followed immediately by at least two lower values was considered as a pulse (Gabai et al., 1994). Data were analysed using the GLM procedure of SAS (SAS, 1996). Dry matter intake, rectal temperature and plasma concentrations of hormones were evaluated utilising two different models. The first model was used to evaluate the differences between groups A and B during the covariate period. It considered the following effects: group (A and B), heifer within group, day (day of OC), group 3 day interaction and the error term. Differences between groups were analysed according to the test of hypotheses using heifer within group as the error term (SAS, 1996). The second model considered the following effects: treatments (TC, HAT and TCRF), heifers, day (day of OC), treatment 3 day interaction and the error term. Least square means were separated with the PDIFF procedure of SAS (SAS, 1996). Significance was set at P , 0.05.

3. Results

3.1. Rectal temperatures and feed intake Rectal temperatures did not differ between groups A and B during the covariate period (38.78C in both groups). The heifers under HAT showed higher (P , 0.01) RT compared with heifers under TC or TCRF (Fig. 2). There were no differences in RT between TC and TCRF treatments (Fig. 2). Dry matter intake (8.861.2 vs. 9.161.6 kg / day / head, P . 0.05) and water intake (37.5262.1 vs. 38.0262.9 l / day / head, P . 0.05) did not differ between groups A and B during the covariate period. Heifers under HAT ingested | 23% less (P , 0.01) DM than heifers under TC (Fig. 3). Water intake was higher (P , 0.01) under HAT than under TC or TCRF (52.8063.3 vs. 38.7062.3 and 34.4062.1 l / day / head, respectively). Significant differences (P , 0.05) in water intake between TC and TCRF treatments were also observed.


Fig. 2. Least square means6S.E. for rectal temperatures (RT) in Holstein heifers kept under thermal comfort (TC d), high ambient temperatures (HAT j) and thermal comfort but restricted fed (TCRF m). *P , 0.05 and **P , 0.01 between HAT and TC or TCRF treatments. No differences were observed between TC and TCRF treatments.

3.2. Hormonal and oestrus cycle changes No differences were observed between groups A and B during the covariate period for mean plasma cortisol concentrations (5.160.5 vs. 4.560.5 ng / ml, P . 0.05, respectively), secretion of progesterone and oestradiol-17b (data not shown), OC length (21.660.5 vs. 21.160.5 days, P . 0.05, respectively), frequency and amplitude of LH and FSH pulses, LH and FSH baseline concentrations (data not shown), mean plasma PRL (61.766.0 vs. 58.167.7 ng / ml, P . 0.05, respectively), frequency and am-

Fig. 3. Least square means6S.E. for dry matter intake (DMI) in Holstein heifers kept under thermal comfort (TC d), high ambient temperatures (HAT j) and thermal comfort but restricted fed (TCRF m). *P , 0.05 and **P , 0.01 between HAT and TC treatments.

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plitude of PRL pulses, and PRL baseline concentrations (data not shown). Before presenting results regarding the hormonal changes during the experimental period, it must be mentioned that two heifers under HAT were diagnosed as having ovarian cysts. The diagnosis was made by ultrasonographic examinations which were carried out for 15 consecutive days for other purposes. The two animals failed to ovulate and presented very low values of plasma LH, FSH and oestradiol-17b. For these reasons, data from the two heifers were not included in the statistical analysis. HAT heifers had lower (P , 0.01) cortisol concentrations, as compared with TC and TCRF heifers (Fig. 4). Significantly higher concentrations of plasma cortisol at pro-oestrus and oestrus were evident, both under HAT and TC, but mean values were always lower (P , 0.05 and P , 0.01 at pro-oestrus and oestrus, respectively) under HAT (Table 1). Mean plasma progesterone concentrations were lower (P , 0.05) under HAT, compared with TC and TCRF treatments (2.4360.06 vs. 4.1560.09 and 4.0060.09 ng / ml, respectively), and a treatment by day interaction was detected (P , 0.01). In particular, heifers under HAT showed lower plasma progesterone (P , 0.05) from days 11 to 17 of OC, compared with TC and TCRF treatments (Fig. 5). No differences were observed between TC and TCRF heifers (Fig. 5).

Fig. 4. Least square means6S.E. for plasma cortisol in Holstein heifers kept under thermal comfort (TC d), high ambient temperatures (HAT j) and thermal comfort but restricted fed (TCRF m). **P , 0.01 between HAT and TC or TCRF treatments. No differences were observed between TC and TCRF treatments.


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Table 1 Least square means6S.E. of plasma oestradiol-17b, luteinizing hormone (LH) and cortisol concentrations in blood samples collected at 3-h intervals for 12 h before and after the LH peak at the third oestrus of the experimental period Hours from LH peak 2 12 29 26 23 0 3 6 9 12

Oestradiol-17b (pg / ml) TC

a

5.1061.12 6.6761.55 6.2661.35 6.2861.26 7.1361.11 5.2261.24 3.6460.40 2.4060.49 1.4260.32

HAT

LH (ng / ml) b

2.6761.44 4.8861.99 5.7161.75 5.8461.67 5.9861.43 5.0361.60 2.3860.52 1.4460.63 1.1560.41

TC

Cortisol (ng / ml) HAT

1.8360.35 1.6960.44 3.5160.90 7.6161.75 18.8561.78 10.6362.77 4.0561.73 1.4060.32 1.1560.20

1.6160.45 2.1760.57 3.3661.16 5.4562.26 17.0762.30 7.4663.57 3.0362.23 1.3060.41 0.9060.25

TC

HAT d

6.1060.63 6.9560.38 d 6.2861.63 d 8.0961.87 D 14.0162.77 D 9.0061.21 D 8.3561.52 D 8.0061.91 D 5.2460.88 D

2.3460.55 c 2.5560.16 c 2.5360.26 c 3.2561.32 C 5.5062.69 C 1.1660.38 C 0.9260.17 C 1.6260.04 C 0.8060.23 C

a

TC, heifers under thermal comfort and fed on an ad libitum basis. HAT, heifers under high ambient temperatures. c,d Means within row with different superscripts within hormone differ (P , 0.05). C,D Means within row with different superscripts within hormone differ (P , 0.01). b

Fig. 5. Least square means6S.E. for plasma concentrations of progesterone from days 0 to 17 of the oestrous cycle in Holstein heifers kept under thermal comfort (TC d), high ambient temperatures (HAT j) and thermal comfort but restricted fed (TCRF m). *P , 0.05 between HAT and TC or TCRF treatments. No differences were observed between TC and TCRF treatments.

No differences were observed in plasma concentrations of oestradiol-17b (2.6560.04, 2.5460.06 and 2.4760.06 pg / ml, for HAT, TC and TCRF, respectively) between the various treatments and neither was the treatment by day interaction significant (Fig. 6). In blood samples collected at 3-h intervals before and after the LH peak in the 3rdO, the concentrations of plasma oestradiol-17b did not vary between TC and HAT heifers (Table 1). No significant differences were observed in OC length between treatments (19.860.4, 20.760.7 and 21.160.4 days (P . 0.05) in HAT (2nd OC), TC

Fig. 6. Least square means6S.E. for plasma concentrations of oestradiol-17b from days 0 to 17 of the oestrous cycle in Holstein heifers kept under thermal comfort (TC d), high ambient temperatures (HAT j) and thermal comfort but restricted fed (TCRF m).

(2nd OC) and TCRF (4th OC), respectively). LH concentrations in samples collected at 3-h intervals during the 3rdO did not differ between TC and HAT heifers (Table 1). Frequency and amplitude of LH and FSH pulses, and LH and FSH baseline concentrations were not significantly different between treatments (Table 2). A marked increase in PRL concentrations (Fig. 7) was observed in heifers under HAT, as compared with their TC contemporaries (198.85610.1 vs. 53.2768.1 ng / ml, respectively; P , 0.01). No differences were detected between TC and TCRF heifers (Fig. 7). The heifers under HAT had higher (P , 0.01) PRL pulse amplitude and


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Table 2 Least square means6S.E. of pulse frequency and amplitude, and baseline concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) in blood collected every 15 min for 12 h during the second (TC and HAT treatments) and fourth (TCRF treatment) oestrus and dioestrus of the experimental period Pulse amplitude a (ng / ml)

Pulse frequency (no. / 12 h) TC LH FSH PRL

Oestrus Dioestrus Oestrus Dioestrus Oestrus Dioestrus

b

6.261.9 3.060.7 2.560.6 1.660.5 4.560.9 f 3.860.9

HAT

c

4.861.1 4.460.9 3.861.0 1.460.2 2.060.7 e 4.060.9

TCRF

d

5.761.2 2.460.5 2.761.3 2.660.7 4.060.4 f 3.060.6

Baseline (ng / ml)

TC

HAT

TCRF

TC

HAT

TCRF

0.960.12 0.960.31 0.760.08 0.760.06 58.966.0 E 50.4611.3 E

0.760.13 0.860.24 0.760.07 0.760.06 231.969.4 F 203.3611.1 G

1.060.11 0.760.27 0.760.09 0.760.07 45.266.0 E 28.265.4 F

1.160.03 0.660.02 0.560.01 0.560.01 36.463.2 E 20.564.0 E

1.060.03 0.660.02 0.560.01 0.560.01 179.263.1 F 133.764.0 F

1.660.04 0.760.02 0.560.01 0.560.01 27.161.3 E 21.162.0 E

a

Pulse amplitude 5 pulse height minus average of all samples. TC, heifers under thermal comfort and fed on an ad libitum basis. c HAT, heifers under high ambient temperatures. d TCRF, heifers restricted fed and kept under thermal comfort. e,f Means within row with different superscripts within hormone differ (P , 0.05). E,F,G Means within row with different superscripts within hormone differ (P , 0.01). b

Fig. 7. Least square means6S.E. for plasma prolactin (PRL) in Holstein heifers kept under thermal comfort (TC d), high ambient temperatures (HAT j) and thermal comfort but restricted fed (TCRF m). *P , 0.05 and **P , 0.01 between HAT and TC or TCRF treatments. No differences were observed between TC and TCRF treatments.

baseline concentrations during OC, compared with their TC and TCRF contemporaries, associated with a decreased (P , 0.05) pulse frequency (Table 2). No differences were observed between TC and TCRF treatments (Table 2).

4. Discussion Rectal temperature values showed that heifers under HAT were indeed heat stressed (Turner, 1982). As indicated in our previous research (Nar-

done et al., 1993) and that of others (Johnson, 1987), the 23% reduction in DM intake observed in HAT heifers can be considered within the expected range of DM intake reduction for the environmental conditions imposed in our trial. Christison and Johnson (1972) reported a decrease in plasma cortisol concentrations in cattle after prolonged exposure to HAT. The reduction in cortisol is related to an adaptation mechanism. Hydrocortisone is a thermogenic hormone, and the reduction of adrenocortical activity under heat stress is considered a thermoregulatory protective action preventing increases in metabolic heat production. Nevertheless, the significant increase in cortisol during pro-oestrus and oestrus, noted under TC and HAT during the 3rdO, suggests that chronic heat stress depresses cortisol concentrations but does not alter the mechanisms responsible for increased secretion during the follicular phase. The depression of cortisol concentrations on the day of oestrus may have a negative effect on the fertility of cattle (Max, 1990). The author reported a lower percentage of cows with palpable ovarian follicles on the day of oestrus and a lower conception rate to first insemination in cows with lower blood concentrations of cortisol on the day of oestrus. It seems that depression of cortisol secretion has a negative effect on ovarian follicle development. As feed restriction had no effects on cortisol secretion, our results also indicate that the depression in cortisol secretion

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observed under HAT is due to heat stress and is not due to a decline in feed intake. Our results regarding the development of ovarian cysts in HAT heifers concur with those reported by Lopez-Diaz and Bosu (1992), who found that stress factors, including heat stress, may be associated with an impairment of reproductive performance that could be related to the development of ovarian cysts. Moreover, the incidence of ovarian cysts under HAT may be associated with the negative effect of heat stress on follicular dynamics during the OC (Stradaioli et al., 1994). Results of previous studies on blood progesterone in cattle kept in a hot environment are conflicting. Our results, a reduction of progesterone observed under HAT, confirm data reported by Howell et al. (1994). Those authors only found lower progesterone concentrations between days 6 and 18 of the OC, during the summer months. As suggested by Wolfenson et al. (1993), the lower progesterone concentrations under HAT might be a consequence of a smaller number of luteal cells. This hypothesis is also supported by other papers (e.g. Younas et al., 1993) which document a reduction in serum progesterone in heat-stressed cows during the luteal phase. A possible explanation for the reduction in progesterone concentrations in cows reared in hot environments might be the lower plasma cholesterol availability and the impairment of lipid metabolism which occurs under such conditions (Ronchi et al., 1999). As is already known, cholesterol represents an indispensable precursor for the de novo synthesis of progesterone (Staples et al., 1998). The alteration of lipid metabolism due to heat stress may, therefore, partly explain the negative effects of HAT on fertility. In contrast, in a recent study, Wilson et al. (1998) observed higher serum concentrations of progesterone in heat-stressed heifers during the luteal phase compared with contemporaries under TC. In the same study, heat stress delayed the regression of the corpus luteum (CL), which explained the slower decline of progesterone observed by the same authors from days 11 to 21 of the OC. Our results regarding the effects of feed restriction on progesterone concur with those reported by several authors. Apgar et al. (1975) studied the effects of a restricted diet (60% of energy requirements) on the sensitivity of the CL to LH in 10 Holstein heifers over four OC

and did not observe any modification in progesterone secretion, even though restricted-fed heifers showed smaller CL compared with ad libitum-fed heifers. More recently, Rhodes et al. (1996) obtained similar results in restricted-fed (for 5 months) Bos Indicus heifers. Those authors observed a reduction in CL dimensions during the OC preceding anoestrus, whereas progesterone concentrations did not differ. Similarly, Burns et al. (1997), using non-lactating beef cows, reported that daily progesterone concentrations did not differ between ad libitum-fed heifers and restricted-fed cows during the OC preceding anoestrus. Contrasting results were reported by Richards et al. (1995), who found a reduction in plasma progesterone concentrations in multiparous cows as a consequence of severe and prolonged feed restriction. In conclusion, our results indicate that the decline in plasma progesterone observed under HAT during the luteal phase should not be ascribed to the reduction in feed intake which also occurred under those conditions. Our findings on oestradiol-17b concur with those reported by Roman-Ponce et al. (1981), but are in contrast with results reported by other authors who observed higher (Rosenberg et al., 1982) or lower (Wilson et al., 1998) oestradiol concentrations in heat-stressed cattle. Such contrasting results are likely to depend on the severity or nature (chronic vs. acute) of heat stress. In the present study, feed restriction did not affect the oestradiol-17b secretory pattern. Our findings confirm those of others (e.g. Richards et al., 1989), indicating that the plane of nutrition does not alter serum oestradiol concentrations. An increase in OC length was observed in cattle exposed to hot environments (Wilson et al., 1998). The absence of effects of heat stress on the concentrations and pattern of secretion of oestradiol-17b observed under HAT in our study may be related to the short-term exposure of the heifers to a hot environment, since the failure of adequate oestradiol secretion precludes the normal sequence of ovarian events. Conflicting results have been reported regarding LH changes in ruminants exposed to hot environments. No changes in the LH secretory pattern in animals exposed to hot environments have been observed in dairy cattle (Gwazdauskas et al., 1981;


Younas et al., 1993). On the contrary, Madan and Johnson (1973) reported a decrease in the LH preovulatory peak in heat-stressed cattle and Gilad et al. (1993) found lower LH basal concentrations and lower LH amplitude in heat-stressed cows with low plasma oestradiol. The absence of significant differences in the LH secretory pattern observed in our study may be related to the relatively short period of time between the beginning of HAT exposure and the LH evaluation. On the basis of these results, it is likely that acute heat stress does not affect LH secretion. As regards the effects of feed restriction on the LH secretory pattern in cattle, in contrast to our findings, other authors (Richards et al., 1989) observed a reduction in LH concentrations and pulse frequency. The main discrepancy between our results and those of other studies may be due to the kind of feed restrictions utilised, being longer and more severe than those applied in our study. Gilad et al. (1993) reported lower concentrations of FSH in acute and chronic heat-stressed cows which also had lower concentrations of oestradiol. The same authors found no alterations in concentrations of FSH in cows exposed to heat stress but which had normal concentrations of oestradiol. The absence of effects of heat stress on FSH secretion in our trial might be related to the short-term exposure of the heifers to the hot environment and to the unaltered concentrations of oestradiol observed in these animals. Feed restriction can affect FSH in several ways. Looper et al. (1996) reported an increase in FSH concentrations after short-term feed restriction and a decrease in FSH after long-term feed deprivation in ovariectomized cows. Gutierrez et al. (1997) found no effects of feed deprivation on FSH concentrations in Hereford 3 Friesian heifers. Our results would indicate that the magnitude (223%) and the length (20 days) of feed restriction imposed in our trial were not severe or long enough to alter normal gonadotrophin secretion. Schams et al. (1980a) found an increase in plasma PRL in heifers exposed to HAT and hypothesised a direct effect of heat stress on PRL concentrations. Considering our findings (no effects of feed restriction) and those of Schams et al. (1980a), we can confirm that the increase in PRL and the changes in the PRL secretory pattern observed under HAT are directly associated with hyperthermia. A possible

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explanation for the marked and prolonged increase in PRL during heat exposure may derive from the role played by PRL in controlling renal hemodynamics (Becker et al., 1985), electrolyte balance (Collier et al., 1982) and water intake (Schams et al., 1980b). Salah et al. (1995) suggested that PRL might have an important role in thermoregulation during exposure to high ambient temperatures. Nevertheless, high plasma concentrations of PRL, even though potentially useful for acclimatisation to hot environments, could be responsible for modifying the resumption of ovarian function in heat-stressed cattle (Weiss et al., 1981).

5. Conclusions The data reported in the present study indicate that heat stress is responsible for significant effects on the reproductive function of Holstein heifers, as assessed by plasma concentrations of progesterone, LH, FSH, PRL and cortisol during the oestrus cycle. Furthermore, our results also indicate that the effects of heat stress on these aspects of the reproductive function are not influenced by a reduction in feed intake which also occurs under high air temperatures. The role of feed restriction in relation to other aspects of reproductive function altered by heat stress and the mechanisms by which heat stress affects reproductive efficiency still need to be clarified.

Acknowledgements The authors thank Prof. M.M. Shafie (Department of Animal Production, Cairo University, Egypt) for his helpful scientific suggestions. The authors are grateful to Prof. S. Raiti (National Hormone and Pituitary Program, NIDDK, Baltimore, MD) for providing bLH (NIH-LH-B10) and bPRL (NIDDKB-PRL-I-1), and antiserum to ovine-FSH (NIDDKanti-oFSH-1), to Dr. D.J. Bolt (USDA Animal Hormone Program, Germplasm Laboratory Beltsville, MD) for providing bFSH (USDA-bFSH-I-2) and to Prof. L. Reichert Jr. for providing bPRL (LER891-bPRL) and bLH (LER 1072-2). Research supported by RAIZ-Mi.P.A., paper no. RZ-247.

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